Pharmaceutical composition comprising an analgesic peptide and method for treating pain

ABSTRACT

A pharmaceutical composition for topical administration comprising an analgesic effective amount of a peptide comprising L-amino acids of the formula (I): pGLU-X—Y-Z M (I) and a pharmaceutically acceptable excipient. X is an amino acid selected from the group consisting of GLY, VAL, GLU, ASP, SER, ALA, ASN, GLN, ILE, LEU, PRO, LYS and ARG, Y is TRP or THR, and Z is any L-amino acid, or Z is null. When Z is any L-amino acid, one but not both of Y and Z is TRP, and when Z is null, Y=TRP. An alkyl group may be attached to an amino acid of the peptide. Also disclosed are the peptide, the preparation of the pharmaceutical composition and a topical method of treating or preventing pain in a mammal.

FIELD OF THE INVENTION

This invention relates to analgesic peptides and their derivatives.

BACKGROUND OF THE INVENTION

Although pain is a crucially important physiological response, it alsoresults in unnecessary suffering and agony. The control and relief ofpain is an important branch of medicine. Pain may come about both as aresult of disease as well as a result of medical treatment such aschemotherapy. In either case, it is important to alleviate the pain asmuch as possible so as to enable the sufferer to function normally.

Two neural pathways relating to pain act concurrently in the body: (1) asensory pathway which senses tissue damage and subsequently produces afeeling of pain; (2) an analgesic pathway which reduces the feeling ofpain and prevents the flow of information about the pain to the centralnervous system (CNS), thus allowing the organism to maintain ifs normalactivity in spite of an injury. Anesthesia can be realized either by useof a drug which inhibits peripheral nerves that act as pain sensors orby enhancement of the natural analgesic system. Since these aredifferent pathways, they are affected by different substances. Forexample, aspirin and lidocaine are active on the peripheral sensorypathway, while morphine and related substances are active on theanalgesic system.

The most efficient analgesics currently in use are morphine-relatedsubstances of opiatic origin. It's well known that the brain makes avariety of endogenic opiates, and this explains the powerful effect ofthese substances. Their action on neurons is mediated by specializedreceptors. Signals regulated by these receptors prevent the flow ofinformation from the peripheral pain neurons to the CNS. These CNSneurons are also sensitive to a variety of other chemical substancesincluding catecholamines (serotonin, noradrenalin etc.), neuroactivepeptides (neurotensin) and inhibitory amino acids (glycin and GABA).

U.S. Pat. No. 4,619,916 to Di Stazio discloses 13 new tripeptides of theformula pGLU-X-TRP, where pGLU is cyclized glutamic acid (pyroglutamicacid) and X may be GLY, VAL, GLU, ASP, SER, ALA, ASN, GLN, ILE, LEU,PRO, LYS and ARG. Also disclosed are a process for their preparation,pharmaceutical formulations containing them for oral or parenteraladministration and their use as hypotensive and analgesic agents.Further disclosed are lower alkyl esters of the tryptophan residue, inparticular methyl or ethyl esters, for use as protecting groups in theproduction of the peptides. The protecting groups are removed at thecompletion of the synthesis process. There is no disclosure of a topicalformulation.

WO 92/19254 discloses α-substituted mono, di, tri, tetra andpentapeptides useful in treating obesity, anxiety, gastrointestinalulcers, pain, stroke and inflammation. Peptides of the formulapGLU-X-TRP are not disclosed.

The following tetrapeptides of the formula pGLU-X-TRP-Z appear in theliterature:

X=L-Ala; Z=L-LeuOH, L-LeuOCH₃, L-LeuNH₂, L-MetOH, L-MetOCH₃, or L-MetNH₂(DE 3,340,208);

X=Lys; Z=L-AlaOH or L-ProOH (Freer, R. J. and Stewart, J. M. (1971)Cienc. Cult. 23(4):539–42; Francis, B. and Kaiser, I. I. (1993) Toxicon31(7):889–899);

X=L-Pro; Z=L-ValNH₂, L-MetOH, L-MetOCH₃, L-MetNH₂ L-MetsulfoxideOH,L-MetsulfoxideOCH₃, or L-MetsulfoxideNH₂ (DE 3,340,208).

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an analgesicpharmaceutical composition which may be administered topically.

It is a further object of the invention to provide novel peptidederivatives.

In a first aspect, the present invention provides a pharmaceuticalcomposition for topical administration comprising an analgesic effectiveamount of a peptide comprising L-amino acids of the formula (I):pGLU-X—Y-Z  (I)

wherein X is an amino acid selected from the group consisting of GLY,VAL, GLU, ASP, SER, ALA, ASN, GLN, ILE, LEU, PRO, LYS and ARG,

Y is TRP or THR,

and Z is any L-amino acid, or Z is null,

and wherein when Z is any L-amino acid, one but not both of Y and Z isTRP, and when Z is null, Y=TRP,

or an analgesic effective amount of a peptide derivative in which analkyl group is attached to an amino acid of the peptide, and apharmaceutically acceptable excipient.

It has now been discovered that certain peptides may be used as anactive ingredient in topical analgesic compositions.

The active ingredient of the composition of the invention is a peptideof the formula (I). Examples of peptides according to the invention aretripeptides and tetrapeptides in which pGLU is the NH₂ terminal aminoacid and TRP is at the third (Y) or fourth (Z) amino acid position.Examples of preferred peptides are pGLU-ASN-TRP—OH (pENW),pGLU-GLU-TRP—OH (pEEW), pGLU-ASN-TRP-THR—OH (pENWT), pGLU-ASN-TBR-TRP—OH(pENTW), and pGLU-ASN-TRP-LYS—OH (pENWK).

A peptide derivative according to the invention is one in which an alkylchain has been attached to the peptide. This can be done by attaching afatty acid to an amine group, for example to the ε-amine group of alysine or arginine residue, thus obtaining an alkyl amide of thepeptide, or to an hydroxyl group, thus obtaining an alkyl ester of thepeptide. The alkyl chain may be attached to any of the amino acids ofthe peptide capable of reacting with the alkyl chain, as is well knownto the skilled man of the art. The alkyl chain may be of any length, butis preferably of medium to long chain length, e.g. 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29 or 30 carbons. Examples of peptide derivatives arepGLU-ASN-TRP-LYS(octanoyl)-OH (pENWK—C8) and pGlu-Asn-Trp-O-octyl(pENW—C8).

An “analgesic effective amount” is an amount of active ingredientcapable of bringing about the desired pharmacological effect, i.e. thereduction or prevention of pain. This amount depends on a number ofparameters such as the exact composition of the active ingredient andcarrier, the location of administration, the source of the pain, etc.The amount can be easily determined by the average skilled man of theart by carrying out a limited amount of dose response experiments, e.g.by applying a range of concentrations of a given formulation to aspecified location on the body. Examples of concentrations that havebeen found to be effective include, but are not limited to, 0.0015–0.02mg/g carrier.

The pharmaceutical composition of the invention is formulated fortopical administration. Such a composition would also comprise one ormore pharmaceutically acceptable carriers or excipients such as amixture of Lanolin and Vaseline for topical use in an ointment, cream orsalve. Other carriers for topical use are well known to the skilled manof the art and are included in the scope of the invention.Fragrance-emitting, stabilizers, colorants, thickening agents and otherconventional substances may be included in the composition.

The invention also provides a use of an analgesic effective amount of apeptide comprising L-amino acids of the formula (I):pGLU-X—Y-Z  (I)

wherein X is an amino acid selected from the group consisting of GLY,VAL, GLU, ASP, SER, ALA, ASN, GLN, ILE, LEU, PRO, LYS and ARG,

Y is TRP or THR,

and Z is any L-amino acid, or Z is null,

and wherein when Z is any L-amino acid, one but not both of Y and Z isTRP, and when Z is null, Y=TRP,

or of an analgesic effective amount of a peptide derivative in which analkyl group is attached to an amino acid of the peptide, in thepreparation of a topical pharmaceutical composition for the treatment orprevention of pain.

A further aspect of the invention is a method of treating or preventingpain in a mammal comprising topically administrating to the mammal ananalgesic effective amount of a peptide comprising L-amino acids of theformula (I):pGLU-X—Y-Z  (I)

wherein X is an amino acid selected from the group consisting of GLY,VAL, GLU, ASP, SER, ALA, ASN, GLN, ILE, LEU, PRO, LYS and ARG,

Y is TRP or THR,

and Z is any L-amino acid, or Z is null,

and wherein when Z is any L-amino acid, one but not both of Y and Z isTRP, and when Z is null, Y=TRP,

or an analgesic effective amount of a peptide derivative in which analkyl group is attached to an amino acid of the peptide.

The topical administration of the peptide may be in a conventionalmanner for topical compositions.

As the composition of the invention sometimes acts after a lag period,it is to be expected that it will be especially effective with respectto chronic pain, although it may be used to treat any type of pain.

In a still further aspect, there is provided a peptide comprisingL-amino acids of the formula (I):pGLU-X—Y-Z  (I)

wherein X is an amino acid selected from the group consisting of GLY,VAL, GLU, ASP, SER, ALA, ASN, GLN, ILE, LEU, PRO, LYS and ARG,

Y is TRP or THR,

and Z is any L-amino acid, or Z is null,

and wherein when Z is any L-amino acid, one but not both of Y and Z isTRP, and when Z is null, Y=TRP,

or a derivative of the peptide in which an alkyl group is attached to anamino acid, wherein the length of the alkyl is C4 or longer,

with the proviso that when Z is any L-amino acid, if X=ALA, Z is not LEUor MET, if X=LYS, Z is not ALA or PRO, and if X=PRO, Z is not VAL orMET,

and with the further proviso that when Z is null, the peptide has thealkyl group attached to an amino acid thereof.

These peptides are unknown in the literature.

A further embodiment of this aspect of the invention is a pharmaceuticalcomposition for treatment or prevention of pain comprising an analgesiceffective amount of the peptide of the invention or of an alkyl ester oramide thereof. The pharmaceutical composition may be administeredorally, parenterally or topically.

DETAILED DESCRIPTION OF EMBODIMENTS

Methods and Materials

Preparation of Peptide and Derivatives

1. Synthesis of pGlu-Asn-Trp-Lys(Octanoyl)-OH

In one embodiment of the invention, the synthesis of the peptide wascarried out manually by a stepwise 9-fluorenylmethoxycarbonyl (Fmoc)solid phase peptide synthesis (SPPS) procedure on Fmoc-Lys(Mtt)-Wangresin (loading of 0.25 mmole on 1 g of preloaded resin).

At the first step the Mtt (4-methyltrityl) protecting group wasselectively removed by treatment with 1% TFA in DCM. Octanoic acid wasattached to the free amino group (via an amide bond) by regular couplingprocedure applying 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) reagent in the presence ofN-hydroxybenzotriazole (HOBt). The same coupling method was applied forthe attachment of other amino acids as well. Completion of the couplingstep was detected by the Kaiser test (a few resin beads from thereaction are washed with ethanol and transferred into a small glasstube; 2 drops of the following solutions are added: ninhydrin 5% inethanol, phenol 80% in ethanol, potassium cyanide 0.00002M in pyridine;the sample is mixed and heated to 120° C. for 4–6 minutes. A positivetest is indicated by blue resin beads). The Fmoc group was then removedby 20% piperidine, and after washing of the resin the second amino acid(Fmoc-Trp(Boc)) was introduced to restart another coupling step.

These steps were repeated each time with an additional amino acidaccording to the peptide sequence. The amino acids used were Fmoc-N^(α)protected. Trifunctional amino acids were side-chain protected asfollows: Trp(Boc), Asn(Trt). Each Fmoc amino was activated in situ usingHBTU/HOBt and subsequently coupled to the resin for 50 minutes.Diisopropylethylamine (DIEA) was used during coupling as an organicbase. The Fmoc protecting group on the α-amine was then removed with 20%piperidine in N-methylpyrrolidone (NMP) for 20 min. Three equivalents ofthe activated amino acids were employed in the coupling reactions. Thedeprotection and coupling steps were repeated with the addition of eachsubsequent amino acid until the peptide synthesis was completed. Thepeptide-resin was washed with NMP, followed by DCM, and dried undervacuum.

This peptide, prepared as described above, was cleaved from the resinusing a 95% TFA, 5% triisopropylsilane (TIS) solution for 2 hours atroom temperature. The product was precipitated by the addition of 10volumes of ether, filtered and dried in vacuum. Typically, 150 mg of thepeptide was obtained from 1 g of peptide-resin. It was identified byLC/MS (M⁺¹=684.7).

Purification

The crude peptide was purified on a preparative RP-HPLC column (C₁₈ 5 μmPhenomenex Luna column, 10 mm I.D.×25 cm). The chromatography was doneunder the following conditions: A=H₂O/TFA 0.05%; B=ACN/TFA 0.05%; λ=214nm; flow=5 ml/min. 50 mg peptide were loaded on the column. A gradientof 15% to 50% B at 45 min was used. Fractions of the main peak werecollected and characterized by analytical HPLC. Best fractions werecombined together and lyophilized to obtain the required product, whichwas >95% pure.

2. Preparation of pGlu-Asn-Trp-O-octyl

Preparation of this peptide may also be carried out via a solutionsynthesis protocol containing following steps:

a. Preparation of Boc-Trp-O-octyl (octyl ester of Boc-Trp)

Boc-Trp (6.1 mmole), octanol (6.8 mmole), DMAP (0.74 g, 6.1 mmole), anddicyclohexylcarbodiimide (DCC) (1.4 g, (6.8 mmole) are introduced to DCM(40 ml) at 0° C. The mixture is stirred and the cooling bath is removedso that the temperature slowly rises to room temperature. The reactionis stirred overnight and then is filtered to remove the dicyclohexylurea(DCU). The solid is washed with DCM and the combined mother liqueur isadjusted to pH=4–5 HCl (0.1 N). The organic phase is washed by water(3×30 ml), dried over MgSO₄, filtered and evaporated to obtain crudeproduct.

b. Removal of Boc Group

Boc-Trp-O-octyl is dissolved in ether at room temperature. HCl (4N) indioxane is introduced (4:1 molar excess) and the reaction mixture isstirred for ca 1 h. Unprotected product precipitates as the HCl salt. Itis filtered, washed with ether and dried.

c. DCC/HOBt Coupling Procedure

Boc protected amino acid (14.2 mmole) in DCM (35 ml) is cooled to 0° C.HOBt.H₂O (12.9 mmole, 1.75 g) and DCC (15.5 mmole, 3.2g) are added to areaction mixture and vigorously stirred for 1 h. Deprotected peptide(after removal of the Boc group) dissolved in DCM (20 ml) and TEA (18.1mmole) is introduced and the mixture is stirred for an additional 30min. at 0° C. The cooling bath is removed and the reaction is leftovernight at room temperature.

Products mixture is evaporated to dryness under reduced pressure andEtOAc (150 ml) is added. Insoluble particles (DCU) are removed byfiltration and mother liquor is washed with brine (2×35 ml), NaHCO₃ (5%;2×35 ml), and water 2×35 ml). The organic phase is dried over MgSO₄, andevaporated under reduced pressure.

At the end of the peptide synthesis (the last stage being the couplingof pGlu) a peptide is obtained as a crude product. It is identified byLC/MS and purified similarly to the first peptide(pGlu-Asu-Trp-Lys(octanoyl)-OH).

3. Purification from Snake Venom

Some of the peptides of the invention may also be isolated from snakevenom, including venom obtained from snakes of the Viperidae, Elapidaeand Crotalidae families. For example, pENW may be purified from Najamelanoleuca venom on a Mono Q column using 20 mM Tris-HCl buffer, pH7.0. The fraction which elutes from the Mono Q column at 12–18 minutes(with a peak at 14.3 minutes) is further purified on HPLC as follows.The fraction is loaded onto an HPLC Spherisorb 5 column, 5μ, 250×4.6 mm,phase Sep. S/N 142110, and eluted using a gradient of 20 mM NH₄Ac (pH5.5) and AcN. In an alternate purification, the venom may be purified onthe Mono Q column using 20 mM ammonium acetate buffer, pH 6.9. Thefraction with a peak at 17 minutes is further purified by HPLC asdescribed above. NMR, HPLC and LC/MS analyses revealed the identity ofthe synthetic and natural peptides.

Similarly, pENW, pEEW and pENTW or pENWT have been purified fromCrotalus adamanteus venom, and pENW has been purified from V. palestinaevenom.

Assays

1. Analgesic Assay

In each test, a few tens of hamsters of similar weight and age wereused. The hamsters were divided into groups according to the number ofsamples to be tested. Ointment (50% Lanolin and 50% Vaseline) containingthe tested substance was applied to the animal's fur on the back region.The fur was not removed so as to ensure that no damage to the skinoccurred. A control group of hamsters was treated with ointment withoutthe fraction of the invention. Hamsters were treated by topicalapplication for 6, 14, 21 or 28 consecutive days. The test foranalgesity was conducted following the last application of the ointment.In an alternate protocol, analgesity was tested following a singleapplication of the ointment.

Subsequently to application of the ointment, the hamsters “clean”themselves by dispersing the ointment all over their body with theirtongue and legs. Thus, some of the ointment is introduced into the oralcavity and possibly also into the intestine of the hamsters.

In a typical test, a constant amount of ointment with or without ananalgesic substance is applied to each animal for a predetermined periodof one or more days. Following this period, pain is induced by asubcutaneous injection of 0.5 ml of 1N HCl/0.1 kg body weight in thefemur region. The hamsters respond to the HCl injection by touching thearea of injection with the tongue, this being called a “lick”. 20minutes after injection the hamster is observed for 60 min and thenumber of “licks” are counted. The number of “licks” serves as aquantitative indication of the HCl induced pain.

The analgesic effect is determined by comparing the mean number of“licks” in control animals to the number in treated animals. Thesignificance of the difference was determined using t-test statistics.

EXAMPLE 1

0.6 mg of pENW, pENWK-C8 or pENWGAT (a dimer of pENW) were dissolved in5 ml of DDW, mixed by an ultrasonic mixer for 2 minutes and thendissolved in 315 gr of ointment. The final peptide concentration was0.002 mg/gr.

6 applications were made over a period of 4 weeks. The test foranalgesity was conducted 10 days following the last application of theointment. The results are summarized in the following table:

active number of mean number Std. ingredient animals of licks Dev. SEMp* control 25 113.9 69.1 13.8 — pENW 27 59.7 58.9 11.3 0.0017 pENWK-C828 37.9 34.8 6.6 <0.0001 pENWGAT 32 117.0 81.2 14.4 0.9872 *Mann-Whitneyrank sum test

It can be clearly seen that the topical application of the tripeptidehad a significant effect on reducing the pain of the animals, and thatthe tetrapeptide derivative was even more effective. The addition of 3amino acids to the C-terminal of the tripeptide completely abolished itsactivity.

EXAMPLE 2

Compositions at a concentration of 0.020 mg/g were prepared as inExample 1 containing the following active ingredients: pENW, pEEW, orpENTW or pENWT. 6 applications were made over a period of 5 weeks. Theresults are summarized below:

active number of mean number ingredient animals of licks SEM p* control24 117 6 — pENW 25 44 31 <0.05 pEEW 26 35 30 <0.05 pENTW or 25 39 26<0.05 pENWT *Dunn's method

It can be seen that all of the assayed peptides have significantanalgesic activity.

EXAMPLE 3

A composition containing pENW—C8 at a concentration of 0.002 mg/gointment was prepared as in Example 1. A single application was made tothe animals, and analgesity was assayed 11, 23 and 48 dayspost-application. Ointment without an active substance was applied tothe control animals.

The results summarized below:

# of days post number of mean number application animals of licks SEM p*control 26 106 11.2 — 11 28 66 10 <0.05 23 27 35.7 6.5 <0.05 48 21 62.411 <0.05 *Dunn's method

The results indicate that the analgesic effect builds over time,reaching a peak after around 23 days, and subsequently declines.

1. A topical pharmaceutical composition, consisting of apharmaceutically acceptable excipient, and an analgesic effective amountof a peptide comprising L-amino acids of the formulapGlu-X—Y-Z  (I),  wherein X is an amino acid selected from the groupconsisting of GLY, VAL, GLU, ASP, SER, ALA, ASN, GLN, ILE, LEU, PRO, LYSand ARG, Y is TRP or THR, and Z is any L-amino acid, or Z is null;wherein Z is any L-amino acid, one but not both of Y and Z is TRP, andwhen Z is null, Y=TRP, or a peptide thereof having a (C₂–C₃₀) alkylgroup attached thereto, wherein the topical pharmaceutical compositionis formulated for topical administration.
 2. The pharmaceuticalcomposition according to claim 1, wherein said alkyl group is attachedby an amide linkage.
 3. The pharmaceutical composition according toclaim 1, wherein said alkyl group is attached by an ester linkage. 4.The pharmaceutical composition according to claim 1, wherein X is ASN.5. The pharmaceutical composition according to claim 1, wherein saidalkyl is selected from the group consisting of C₄–C₃₀.
 6. Thepharmaceutical composition according to claim 5, wherein said alkyl isan octyl group (C₈).
 7. The pharmaceutical composition according toclaim 1, wherein said peptide is a tetrapeptide.
 8. The pharmaceuticalcomposition according to claim 1, wherein said peptide is a tripeptideand Z is null.
 9. A method of treating pain, comprising topicallyadministering to a subject in need thereof an analgesic effective amountof a peptide consisting of L-amino acids of the formulapGIu-X—Y-Z  (I)  wherein X is an amino acid selected from the groupconsisting of GLY, VAL, GLU, ASP, SER, ALA, ASN, GLN, ILE, LEU, PRO, LYSand ARG, Y is TRP or THR, and Z is any L-amino acid, or Z is null;wherein Z is any L-amino acid, one but not both of Y and Z is TRP, andwhen Z is null, Y=TRP; or a peptide thereof having an alkyl groupattached thereto.
 10. The method according to claim 9, wherein saidalkyl group is attached by an amide linkage.
 11. The method according toclaim 9, wherein said alkyl group is attached by an ester linkage. 12.The method according to claim 9, wherein X is ASN.
 13. The methodaccording to claim 9, wherein said alkyl is selected from the groupconsisting of C₄–C₃₀.
 14. The method according to claim 13, wherein saidalkyl is an octyl group (C₈).
 15. The method according to claim 9,wherein said peptide is a tetrapeptide.
 16. The method according toclaim 9, wherein said peptide is a tripeptide and Z is null.
 17. Amethod of treating pain, comprising topically administering to a mammalan analgesic effective amount of a peptide consisting of L-amino acidsof chemical formulapGlu-X—Y-Z  (I)  wherein X is an amino acid selected from the groupconsisting of GLY, VAL, GLU, ASP, SER, ALA, ASN, GLN, ILE, LEU, PRO, LYSand ARG, Y is TRP or THR, and Z is any L-amino acid, or Z is null;wherein Z is any L-amino acid,  one but not both of Y and Z is TRP, andwhen Z is null, Y=TRP; or a peptide thereof having an alkyl groupattached thereto.
 18. The method according to claim 17, wherein saidalkyl group is attached by an amide linkage.
 19. The method according toclaim 17, wherein said alkyl group is attached by an ester linkage. 20.The method according to claim 17, wherein X is ASN.
 21. The methodaccording to claim 17, wherein said alkyl is selected from the groupconsisting of C₄–C₃₀.
 22. The method according to claim 21, wherein saidalkyl is an octyl group (C₈).
 23. The method according to claim 17,wherein said peptide is a tetrapeptide.
 24. The method according toclaim 17, wherein said peptide is a tripeptide and Z is null.
 25. Apeptide comprising L-amino acids of chemical formulapGlu-X—Y-Z  (I)  wherein X is an amino acid selected from the groupconsisting of GLY, VAL, GLU, ASP, SER, ALA, ASN, GLN, ILE, LEU, PRO, LYSand ARG, Y is TRP or THR, and Z is any L-amino acid; wherein one but notboth of Y and Z is TRP; or a peptide thereof having a (C₅–C₃₀) alkylgroup attached thereto, wherein X and Y are as defined hereinabove, andZ is any L-amino acid, or Z is null; wherein when Z is any L-amino acid,if X=ALA, Z is neither LEU nor MET, if X=LYS, Z is neither ALA nor PRO,and if X=PRO, Z is neither VAL nor MET.
 26. The peptide according toclaim 25, wherein said alkyl is selected from the group consisting ofC₅–C₃₀.
 27. The alkyl ester according to claim 26, wherein said alkyl isan octyl group (C₈).
 28. A tetrapeptide according to claim
 25. 29. Atripeptide according to claim
 25. 30. A pharmaceutical composition fortreatment of pain, comprising an analgesic effective amount of thepeptide according to claim
 25. 31. A pharmaceutical composition fortreatment or prevention of pain, comprising an analgesic effectiveamount of the alkyl ester or alkyl amide of a peptide according to claim25.
 32. The method according to claim 9, wherein said peptide ispGlu-Asn-Trp-Thr, pGlu-Asn-Thr-Trp, or pGlu-Asn-Trp-Lys-C₈.
 33. Thepharmaceutical composition according to claim 1, wherein said peptide ispGlu-Asn-Trp-Thr, pGlu-Asn-Thr-Trp, or pGlu-Asn-Trp-Lys-C₈.
 34. Thepharmaceutical composition according to claim 25, wherein said peptideis pGlu-Asn-Trp-Thr, pGlu-Asn-Thr-Trp, or pGlu-Asn-Trp-Lys-C₈.